Method for detecting nucleic acid of target microorganism from sample collected from skin using corynebacterium spp. or cutibacterium spp. as endogenous internal control

Abstract

The present invention relates to a method for detecting a nucleic acid of a target microorganism from a sample collected from skin by using a nucleic acid of Corynebacterium spp. or Cutibacterium spp. as an endogenous internal control nucleic acid, and a composition for nucleic acid amplification used in the method. The endogenous internal control according to the present invention is present in the sample from the beginning, thereby eliminating the inconvenience of having to add same separately after sample collection, and can be used as an internal control for the sample collection process, an internal control for the nucleic acid extraction process, and an internal control for the nucleic acid amplification process. In addition, by using the nucleic acid of Corynebacterium spp. or Cutibacterium spp. as an endogenous internal control, it is possible to detect the presence or absence of a target microbial nucleic acid in a sample collected from skin with high accuracy by minimizing false negative and false positive determinations.

Description

A method for detecting nucleic acids of target microorganisms from a sample collected from the skin using CORYNEBACTERIUM SPP. or CUTIBACTERIUM SPP. as an internal control.

[0001] Cross-reference to related applications

[0002] This patent application claims priority to Republic of Korea Patent Application No. 2024-0084882, filed with the Korean Intellectual Property Office on June 27, 2024, the disclosures of which are incorporated herein by reference.

[0003] Technology field

[0004] The present invention relates to a method for detecting a nucleic acid of a target microorganism from a sample collected from the skin using a nucleic acid of Corynebacterium spp. or Cutibacterium spp. as an endogenous internal control nucleic acid, and to a composition for amplifying a nucleic acid used in the method.

[0005]

[0006] Human diseases can be detected through in vitro and in vitro diagnostics. In vitro diagnostics analyze the cause of disease through X-rays, CT scans, and other methods, while in vitro diagnostics analyze the cause of disease through urine, blood, tissue cells, and other methods.

[0007] In vitro diagnostics include immunochemical diagnostics, self-blood glucose monitoring, point-of-care diagnostics, and molecular diagnostics. Among these, molecular diagnostics directly examines genes using techniques like polymerase chain reaction (PCR). This involves extracting nucleic acids containing the pathogen's genetic information from samples such as saliva, blood, or stool from infected individuals, then amplifying these nucleic acids to confirm the presence of disease. This type of molecular diagnostics offers greater accuracy than blood or urine tests and eliminates the need for tissue biopsies, enabling early diagnosis, prevention, and effective treatment.

[0008] In molecular diagnostics, accurate and appropriate sample collection is essential for accurate test results. Insufficient sample volume or contamination during sample collection can lead to inaccurate test results, increasing retest rates and delaying test result reporting. Furthermore, as described above, molecular diagnostics involves extracting and amplifying nucleic acids from a sample. If loss occurs during the nucleic acid extraction process or if the sample solution contains substances that inhibit the amplification reaction (e.g., heparin, detergents, protein denaturants, or organic solvents), amplification efficiency will be reduced, preventing sufficient amplification of pathogen nucleic acids. In such cases, despite the presence of pathogens in the sample, the inability to amplify pathogen nucleic acids can result in false negative results. Furthermore, even when using the same sample, different results can be obtained depending on the degree of loss during the nucleic acid extraction process.

[0009] To address these issues, a method for identifying false negatives using an internal control during the process of extracting nucleic acids from a sample and amplifying the extracted nucleic acids has been developed (U.S. Patent No. 5,770,360). By using a nucleic acid that is either always present in the sample or added to the sample prior to nucleic acid extraction as an internal control, the control nucleic acid is always extracted, amplified, and detected, regardless of the presence or absence of pathogens in the sample. Failure to detect the nucleic acid in the internal control confirms that the false negative occurred due to loss during the extraction process or amplification degradation caused by a PCR inhibitor.

[0010] The present inventors have confirmed that HBB and HuBac, which are present in human cells and have been used as internal controls, have low detection rates when detecting target microorganisms from samples collected from human skin, and thus cannot be used as internal controls. Therefore, they recognized the need to develop a new internal control.

[0011]

[0012] Numerous references and patents are cited and cited throughout this specification. The disclosures of these references and patents are incorporated herein by reference in their entirety to further clarify the state of the art and the scope of the present invention.

[0013]

[0014] The present inventors have diligently studied and endeavored to develop a novel internal control that can improve the accuracy of detection by minimizing false negative and false positive judgments in a method for detecting target microorganisms from samples collected from the skin using nucleic acid amplification. As a result, the present inventors have experimentally demonstrated that nucleic acids of Corynebacterium spp. or Cutibacterium spp. can be successfully used as an internal control for the sample collection process, the nucleic acid extraction process, and / or the nucleic acid amplification process of the extracted nucleic acid in a method for detecting target microorganisms from samples collected from the skin using nucleic acid amplification, thereby completing the present invention.

[0015]

[0016] Accordingly, the purpose of the present invention is to provide a method for detecting a nucleic acid of a target microorganism from a sample collected from the skin using a nucleic acid of Corynebacterium spp. or Cutibacterium spp. as an endogenous internal control nucleic acid.

[0017] Another object of the present invention is to provide a composition for amplifying a nucleic acid of a target microorganism from a sample collected from the skin using a nucleic acid of Corynebacterium spp. or Cutibacterium spp. as an endogenous internal control nucleic acid.

[0018]

[0019] Other objects and advantages of the present invention will become more apparent from the detailed description, claims and drawings below.

[0020] According to one aspect of the present invention, the present invention provides a method for detecting a nucleic acid of a target microorganism from a sample collected from the skin using a bacterial nucleic acid selected from the normal skin flora as an endogenous internal control nucleic acid, comprising the following steps:

[0021] (a) A step of collecting and preparing a sample from the skin;

[0022] (b) a step of performing an amplification reaction of nucleic acid in the sample using (i) a pair of primers for amplifying nucleic acid of a target microorganism; and (ii) a pair of primers for amplifying nucleic acid of a bacteria selected from the normal skin flora, which is an endogenous internal control nucleic acid;

[0023] (c) a step of detecting the result of the amplification reaction; and

[0024] (d) determining the validity of the amplification reaction of the target microorganism nucleic acid from the result of the amplification reaction of the internal control nucleic acid; and (i) determining whether the nucleic acid of the target microorganism is present in the sample based on the determined validity and (ii) the result of the amplification reaction of the target microorganism nucleic acid.

[0025] The present inventors have conducted extensive research efforts to develop a novel internal control that can be used in a method for detecting target microorganisms from skin samples using nucleic acid amplification. As a result, a novel protocol for detecting target microorganism nucleic acids from skin samples was established using bacterial nucleic acids selected from the normal skin flora as internal control nucleic acids. According to this novel protocol, bacterial nucleic acids selected from the normal skin flora can be used as internal control nucleic acids for the sample collection process, the nucleic acid extraction process, and / or the nucleic acid amplification process of the extracted nucleic acids.

[0026] The term “normal skin flora” as used herein means the entire community of bacteria formed on the skin of a human or animal.

[0027] According to one embodiment of the present invention, the normal skin flora is the normal skin flora of human skin.

[0028] According to one embodiment of the present invention, the normal skin flora may include, but is not limited to, Corynebacterium, Cutibacterium, Staphylococcus, Enhydrobacter, and Micrococcus. More specifically, the normal flora of the skin includes Corynebacterium spp., Cutibacterium spp., Staphylococcus spp., Enhydrobacter spp., and Micrococcus spp., and more specifically, Corynebacterium tuberculostearicum, Corynebacterium simulans, Corynebacterium afermentans, Corynebacterium fastidiosum, Corynebacterium resistens, Cutibacterium acnes, Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus hominis, Staphylococcus haemolyticus, Staphylococcus warneri, Enhydrobacter aerosaccus, and Micrococcus luteus, and even more specifically, Corynebacterium tuberculostearicum, Corynebacterium simulans, Corynebacterium afermentans, Corynebacterium fastidiosum, Cutibacterium acnes, Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus hominis, Enhydrobacter aerosaccus, and Micrococcus luteus.

[0029] According to another aspect of the present invention, the present invention provides a method for detecting a nucleic acid of a target microorganism from a sample collected from the skin using a nucleic acid of Corynebacterium spp. or Cutibacterium spp. as an endogenous internal control nucleic acid, comprising the following steps:

[0030] (a) A step of collecting and preparing a sample from the skin;

[0031] (b) a step of performing an amplification reaction of nucleic acid in the sample using (i) a pair of primers for amplifying nucleic acid of a target microorganism; and (ii) a pair of primers for amplifying nucleic acid of Corynebacterium spp. or Cutibacterium spp., which is an endogenous internal control nucleic acid;

[0032] (c) a step of detecting the result of the amplification reaction; and

[0033] (d) determining the validity of the amplification reaction of the target microorganism nucleic acid from the result of the amplification reaction of the internal control nucleic acid; and (i) determining whether the nucleic acid of the target microorganism is present in the sample based on the determined validity and (ii) the result of the amplification reaction of the target microorganism nucleic acid.

[0034] The present inventors have made extensive research efforts to develop a novel internal control that can be used in a method for detecting target microorganisms from skin samples using nucleic acid amplification. As a result, a novel protocol for detecting target microorganism nucleic acids from skin samples using nucleic acids of Corynebacterium spp. or Cutibacterium spp. as an internal control nucleic acid has been established. According to this novel protocol, the nucleic acids of Corynebacterium spp. or Cutibacterium spp. can be used as an internal control for the steps of collecting a sample, extracting nucleic acids from a sample, and / or amplifying the extracted nucleic acids.

[0035] According to one embodiment of the present invention, the Corynebacterium spp. and Cutibacterium spp. are normal skin flora.

[0036]

[0037] Figure 1 is a flowchart of processes for implementing the method of the present invention according to one embodiment of the present invention. The method of the present invention is described with reference to Figure 1 as follows:

[0038] Step (a): Collect and prepare a sample from the skin (110)

[0039] First, the method of the present invention includes the step of (a) collecting and preparing a sample from the skin.

[0040] The term "sample" in this specification means a sample obtained from a human or animal subject for which nucleic acid of a target microorganism is to be detected from a sample collected from the skin by a nucleic acid amplification method.

[0041] According to one embodiment of the present invention, the sample of step (a) is collected from moist sites, dry sites, or oily sites of the skin. More specifically, the sample of step (a) is collected from a moist site of the skin, and even more specifically, the moist site of the skin is the armpit, the antecubital fossa, the navel, the groin, the popliteal fossa, or the sole of the foot.

[0042] According to one embodiment of the present invention, the sample is a sample obtained from a test subject of a human or animal suspected of being infected with a pathogenic microorganism.

[0043] According to one embodiment of the present invention, the animals include, but are not limited to, primates, livestock (e.g., pigs, sheep, cows, horses, and donkeys), laboratory animals (e.g., rats, mice, guinea pigs, hamsters, and rabbits), pet animals (e.g., dogs and cats), farmed wild animals (e.g., squirrels, foxes, kangaroos, and deer), and birds.

[0044] According to one embodiment of the present invention, nucleic acids in a sample can be directly analyzed without a step of extracting nucleic acids from the sample. For example, the methods disclosed in Pannacio et al. (Nucleic Acids Res. 1993 September 25; 21(19): 4656) and Pandori et al. (BMC Infect Dis. 2006 June 24; 6: 104) can be used.

[0045] According to one embodiment of the present invention, the preparation of the sample further comprises a step of extracting nucleic acid from the sample.

[0046] The term "nucleic acid" or "nucleic acid molecule" as used herein refers to a deoxyribonucleotide or ribonucleotide polymer in single-stranded or double-stranded form, wherein the nucleotides include derivatives of natural nucleotides, non-natural nucleotides, or modified nucleotides that can function in the same manner as naturally occurring nucleotides.

[0047] Nucleic acids can be extracted from the above samples using various methods known in the art, and specific methods thereof are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001). In addition, various nucleic acid extraction kits are commercially available depending on the type of sample, and those skilled in the art can extract nucleic acids from various samples using commercially available kits.

[0048]

[0049] Step (b): Performing an amplification reaction of nucleic acids in the sample (120)

[0050] Next, the method of the present invention includes a step of performing an amplification reaction of the nucleic acid in the sample using (i) a pair of primers for amplifying the nucleic acid of the target microorganism; and (ii) a pair of primers for amplifying the nucleic acid of Corynebacterium spp. or Cutibacterium spp., which is an endogenous internal control nucleic acid.

[0051] The amplification reaction of the nucleic acid may additionally include (i) a probe for detecting a nucleic acid of the target microorganism; and (ii) a probe for detecting a nucleic acid of the Corynebacterium spp. or Cutibacterium spp.

[0052] The term "primer" as used herein means an oligonucleotide that acts as an initiation point for synthesis under conditions that induce the synthesis of a primer extension product complementary to a nucleic acid chain (template), i.e., the presence of nucleotides and a polymerizing agent such as a nucleic acid polymerase, and conditions of suitable temperature and pH.

[0053] The term "probe" as used herein means a single-stranded nucleic acid molecule comprising a portion or portions substantially complementary to a target nucleic acid sequence.

[0054] The primers or probes used in the present invention may include natural NMPs (i.e., AMP, GMP, CMP, and UMP), natural dNMPs (i.e., dAMP, dGMP, dCMP, and dTMP), modified nucleotides, or non-natural nucleotides.

[0055] The primer must be sufficiently long to prime the synthesis of the extension product in the presence of the polymerizing agent. The appropriate primer length depends on several factors, such as temperature, application, and primer source.

[0056] The term "annealing" or "priming" herein refers to the juxtaposition of an oligonucleotide or nucleic acid to a template nucleic acid, which juxtaposition causes a polymerase to polymerize the nucleotides to form a nucleic acid molecule complementary to the template nucleic acid or a portion thereof.

[0057] The term "complementary" as used herein means sufficiently complementary that a primer or probe selectively hybridizes to a target nucleic acid molecule under given annealing or hybridization conditions, and includes both substantially complementary and perfectly complementary, and specifically means perfectly complementary.

[0058] As used herein, the term “target nucleic acid,” “target nucleic acid sequence,” or “target nucleic acid molecule” refers to a nucleic acid molecule that is ultimately to be amplified or detected, and is annealed or hybridized with a primer under specific hybridization conditions.

[0059] The term "target microorganism" used in the present invention refers to a microorganism to be detected in a sample collected from the skin.

[0060] The term "nucleic acid of target microorganism" used in the present invention means the nucleic acid of a microorganism to be detected in a sample collected from the skin.

[0061] According to one embodiment of the present invention, the target microorganism to be detected through the method of the present invention refers to a microorganism present in a sample collected from the skin of a human or animal, and the microorganism may include, but is not limited to, bacteria, yeast, fungi, viruses, protozoans, etc.

[0062] According to one embodiment of the present invention, the target microorganism is a drug-resistant microorganism, a dermatophyte, or methicillin-resistant staphylococcus aureus (MRSA).

[0063] According to a specific embodiment of the present invention, the drug-resistant microorganism is a multidrug resistant organism, and more specifically includes Acinetobacter baumannii, Pseudomonas aeruginosa, Candida auris, and Stenotrophomonas maltophilia.

[0064] The nucleic acid of the target microorganism may include, but is not limited to, a DNA molecule or an RNA molecule.

[0065] According to one embodiment of the present invention, the nucleic acid of the target microorganism may include a resistance gene that confers drug resistance, and may include, for example, a mutation of a gene, a resistance gene mediated by a plasmid or a transposon.

[0066] According to one embodiment of the present invention, the nucleic acid of Corynebacterium spp. or Cutibacterium spp. used as the internal control nucleic acid is different from the nucleic acid of the target microorganism to be detected according to the method of the present invention.

[0067] According to one embodiment of the present invention, the method for detecting nucleic acids of target microorganisms according to the present invention can detect 1 to 30, specifically 1 to 25, 1 to 20, 1 to 15, 1 to 10, or 1 to 5 target microorganism nucleic acids simultaneously, and more specifically, can detect 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 target microorganism nucleic acids, but is not limited thereto.

[0068] According to one embodiment of the present invention, the nucleic acid amplification reaction may use 1 to 30 pairs, specifically 1 to 25 pairs, 1 to 20 pairs, 1 to 15 pairs, 1 to 10 pairs or 1 to 5 pairs of primer pairs for amplification of target microbial nucleic acids, and more specifically, 1 pair, 2 pairs, 3 pairs, 4 pairs, 5 pairs, 6 pairs, 7 pairs, 8 pairs, 9 pairs, 10 pairs, 15 pairs, 20 pairs, 25 pairs or 30 pairs of primer pairs for amplification of target microbial nucleic acids, but is not limited thereto.

[0069] The term "hybridization" as used herein refers to the formation of a double-stranded polynucleotide through non-covalent bonding between complementary nucleotide sequences of two single-stranded polynucleotides under certain hybridization conditions or stringent conditions.

[0070] Hybridization can occur when two nucleic acid sequences have perfect complementarity at the site of hybridization (the site where the double-stranded DNA is formed) or when there is a mismatch (e.g., a mismatch of 1 to 4 bases). The degree of complementarity required for hybridization can vary depending on the conditions of the hybridization reaction, particularly temperature.

[0071] In this specification, the terms “hybridization” and “annealing” are not different and are used interchangeably in this specification.

[0072] Amplification of target nucleic acid molecules can be performed using various primer-assisted nucleic acid amplification methods known in the art. Specifically, target nucleic acid amplification is performed using polymerase chain reaction (PCR), a PCR method disclosed in U.S. Patent Nos. 4,683,195, 4,683,202, and 4,800,159. Other examples include ligase chain reaction (LCR) (U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)), strand displacement amplification (SDA) (Walker, et al. Nucleic Acids Res. 20(7):1691-6 (1992); Walker PCR Methods Appl 3(1):1-6 (1993)), transcription-mediated amplification (Phyffer, et al., J. Clin. Microbiol. 34:834-841 (1996); Vuorinen, et al., J. Clin. Microbiol. 33:1856-1859 (1995)), These include nucleic acid sequence-based amplification (NASBA) (Compton, Nature 350(6313):91-2 (1991)), rolling circle amplification (RCA) (Lisby, Mol. Biotechnol. 12(1):75-99 (1999); Hatch et al., Genet. Anal. 15(2):35-40 (1999)), and Q-beta replicase (Lizardi et al., BiolTechnology 6:1197 (1988)).

[0073] A variety of DNA polymerases can be used for the nucleic acid amplification of the present invention, including E. coli DNA polymerase I, thermostable DNA polymerase, and bacteriophage T7 DNA polymerase. Specifically, the DNA polymerase is a thermostable DNA polymerase obtainable from various bacterial species, including Thermus aquaticus (Taq), Thermus thermophilus, Thermus filiformis, Thermus flavus, Thermus antranikianii, Thermus caldophilus, Thermus chliarophilus, Thermus igniterrae, Thermus lacteus, Thermus oshimai, Thermus ruber, Thermus rubens, Thermus scotoductus, Thermus silvanus, Thermus speciesZ05, and Thermus species sps17.

[0074] According to one embodiment of the present invention, amplification of the nucleic acid can be performed by performing PCR.

[0075] According to another embodiment of the present invention, the amplification reaction can be performed by a fast PCR method.

[0076] The term "fast PCR" used herein refers to a PCR method that increases the speed of PCR compared to a general PCR method. The fast PCR can be achieved by controlling various factors such as the extension rate of the DNA polymerase, the temperature change rate (ramp speed) of the thermal cycler, and the complexity of the template. For example, instead of the Taq DNA polymerase used in general PCR, which has a standard extension rate of 1 kb per minute, a Taq DNA polymerase with a standard extension rate of 2-4 kb per minute can be used to achieve fast PCR.

[0077] According to one embodiment of the present invention, the nucleic acid of the Corynebacterium spp. and Cutibacterium spp. may include a nucleotide sequence encoding 16s rRNA or gyrA (DNA gyrase subunit A).

[0078] According to a more specific embodiment of the present invention, the nucleic acid of the Corynebacterium spp. may include a nucleotide sequence encoding 16s rRNA, or the nucleic acid of the Cutibacterium spp. may include a nucleotide sequence encoding gyrA (DNA gyrase subunit A).

[0079] According to another more specific embodiment of the present invention, the nucleic acid of the Corynebacterium spp. comprises the sequence 1 in the sequence listing, or a complementary sequence thereof, or the nucleic acid of the Cutibacterium spp. comprises the sequence 6 in the sequence listing, or a complementary sequence thereof.

[0080] According to one embodiment of the present invention, the Corynebacterium spp. is Corynebacterium tuberculostearicum, Corynebacterium simulans, Corynebacterium afermentans, Corynebacterium fastidiosum, or Corynebacterium resistens, or the Cutibacterium spp. is Cutibacterium acnes, and more specifically, the Corynebacterium spp. is Corynebacterium tuberculostearicum, or the Cutibacterium spp. is Cutibacterium acnes.

[0081] As a specific example, the 16s rRNA gene sequence of Corynebacterium tuberculostearicum can be found in GenBank Accession No. X84247.1, etc., and the gyrA gene sequence of Cutibacterium acnes can be found in GenBank Accession No. KX650495.1, etc.

[0082] According to one embodiment of the present invention, the nucleic acid of Corynebacterium spp. or Cutibacterium spp. can be used as an internal control for (i) the step of collecting a sample, (ii) the step of extracting nucleic acid from the sample, and / or (iii) the step of amplifying the extracted nucleic acid.

[0083] Based on the fact that the above Corynebacterium spp. or Cutibacterium spp. is present on the skin, the present inventors devised a method for detecting the nucleic acid of a target microorganism in a sample collected from the skin using the nucleic acid of Corynebacterium spp. or Cutibacterium spp. as an endogenous internal control nucleic acid.

[0084] In the present invention, the nucleic acid of Corynebacterium spp. or Cutibacterium spp. is amplified simultaneously with the amplification of the target microbial nucleic acid from a sample collected from the skin in the same reaction vessel. At this time, if the amplification of the nucleic acid of Corynebacterium spp. or Cutibacterium spp. is not detected, it may indicate that the desired process did not occur normally in at least one of the steps of collecting the sample, extracting the nucleic acid, and amplifying the nucleic acid in the sample. Therefore, the nucleic acid of Corynebacterium spp. or Cutibacterium spp. can be used as an internal control in each of the above steps. The role of the internal control in each step is described in detail as follows.

[0085] (i) Internal control at the sample collection stage:

[0086] If the sample is not collected properly during the sampling process, for example, if the amount of sample collected is too small to detect nucleic acids, the nucleic acids of the target microorganism may not be detected even if they are present in the sample collected from the skin. This may ultimately result in a false negative result, and such false negative results can be determined by confirming the presence or absence of nucleic acids of the Corynebacterium spp. or Cutibacterium spp. as an internal control.

[0087] (ii) Internal control at the nucleic acid extraction step:

[0088] If nucleic acid loss occurs during the nucleic acid extraction step, i.e., if the amount of nucleic acid extracted as a template for the amplification reaction is insufficient, the efficiency of the amplification reaction may be reduced, resulting in undetectable nucleic acid and false negative results. In such cases, the presence or absence of nucleic acid from the aforementioned Corynebacterium spp. or Cutibacterium spp. can be determined by using an internal control to determine whether a false negative result has occurred.

[0089] (iii) Internal control in the nucleic acid amplification step:

[0090] During the nucleic acid amplification process, if the amplification reaction solution contains a substance that inhibits the amplification reaction (e.g., heparin, surfactant, protein denaturant, organic solvent, etc.), the amplification efficiency may be reduced despite the presence of the target nucleic acid, resulting in a false negative result without detecting the nucleic acid amplification. In such cases, the presence or absence of a nucleic acid of Corynebacterium spp. or Cutibacterium spp. can be determined by confirming the presence or absence of the nucleic acid of the above-mentioned Corynebacterium spp. or Cutibacterium spp. as an internal control.

[0091] According to one embodiment of the present invention, the method according to the present invention further comprises a step of amplifying nucleic acid in the collected sample using a pair of primers for amplifying nucleic acid of the Corynebacterium spp. or Cutibacterium spp., which is an endogenous internal control nucleic acid, thereby determining whether the collection of the sample is valid or invalid.

[0092] According to one embodiment of the present invention, the method according to the present invention further comprises a step of amplifying nucleic acid extracted from the sample using a pair of primers for amplifying nucleic acid of the Corynebacterium spp. or Cutibacterium spp., which is an endogenous internal control nucleic acid, thereby determining whether the extraction is valid or invalid.

[0093] According to one embodiment of the present invention, the amplification reaction of a nucleic acid is carried out in the presence of a label or labeled oligonucleotide (labeled primer or labeled probe) capable of providing a signal dependent on the presence of the nucleic acid to be detected.

[0094] According to one embodiment of the present invention, a signal may be provided from a label during the process of amplifying a nucleic acid to be detected, or a signal may be provided after amplification is completed.

[0095]

[0096] Step (c): Detecting the results of the amplification reaction (130)

[0097] Next, the method of the present invention includes the step of (c) detecting the result of the amplification reaction.

[0098] According to one embodiment of the present invention, the result of a nucleic acid amplification reaction can be detected during the nucleic acid amplification process or after the nucleic acid amplification reaction is completed.

[0099] According to one embodiment of the present invention, detection of the amplification reaction result can be performed by a post-amplification detection method or a real-time detection method.

[0100] The above post-amplification detection method is a method for detecting amplification products after nucleic acid amplification. Post-amplification detection methods include, but are not limited to, methods for separating amplification products by size difference (e.g., electrophoresis) or by immobilizing the amplification products.

[0101] In addition, the above post-amplification detection method may use a post-PCR melting analysis (US Patent No. 5,871,908, US Patent No. 6,174,670 and WO 2012 / 096523) that monitors the fluorescence intensity while raising or lowering the temperature at a certain interval after amplification of the target nucleic acid sequence and then detects the amplification product by the melting profile.

[0102] The above real-time detection method is a method capable of detecting a target nucleic acid sequence while monitoring the amplification of the target nucleic acid in real time.

[0103] The above post-amplification detection method or real-time detection method may use a label or labeled oligonucleotide that provides a signal dependent on the presence of the nucleic acid to be detected.

[0104] According to one embodiment of the present invention, detection of an amplified nucleic acid can be performed by detecting a signal provided from a label during the process of amplifying the nucleic acid to be detected or by detecting a signal provided after amplification of the nucleic acid to be detected is completed.

[0105] For example, this can be done using a non-specific fluorescent dye that non-specifically intercalates into a duplex, which is an amplicon of the target nucleic acid sequence.

[0106] Additionally, labeled primers or labeled probes that specifically hybridize to the target nucleic acid sequence can be used.

[0107] Examples of methods using labeled primers include the Sunrise primer method (Nazarenko et al, 2516-2521 Nucleic Acids Research, 1997, v.25 no.12, and U.S. Pat. No. 6,117,635), the Scorpion primer method (Whitcombe et al, 804-807, Nature Biotechnology v.17 AUGUST 1999, and U.S. Pat. No. 6,326,145), and the TSG primer method (WO 2011 / 078441).

[0108] Examples of methods using labeled probes include, but are not limited to, the molecular beacon method using dual-labeled probes forming a hairpin structure (Tyagi et al, Nature Biotechnology v. 14 MARCH 1996), the hybridization probe method using two single-labeled probes as a donor or acceptor (Bernad et al, 147-148 Clin Chem 2000; 46), the Lux method using single-labeled oligonucleotides (U.S. Pat. No. 7,537,886), and the TaqMan method using not only hybridization of dual-labeled probes but also cleavage of the dual-labeled probes by the 5'-nuclease activity of DNA polymerase (U.S. Pat. Nos. 5,210,015 and 5,538,848).

[0109] Additionally, it can be performed using a dimer formed dependently on the presence of a target nucleic acid sequence. The dimer formed dependently on the presence of the target nucleic acid sequence is not the amplification product of the target sequence itself formed by the amplification reaction, but is a dimer whose amount increases in proportion to the amplification of the target nucleic acid sequence. Dimers formed dependently on the presence of a target nucleic acid sequence can be obtained by various methods, for example, Invader assay (U.S. Patent Nos. 5,691,142, 6,358,691 and 6,194,149), PTO Cleavage and Extension (PTOCE) method (WO 2012 / 096523), PCEC (PTO Cleavage and Extension-dependent Cleavage) method (WO 2012 / 134195), PCE-SH (PTO Cleavage and Extension-dependent Signaling Oligonucleotide Hybridization) method (WO 2013 / 115442), PCE-SC (PTO Cleavage and Extension-dependent Signaling Oligonucleotide Cleavage) method (WO 2013 / 157821), PCE-NH (PTO Cleavage and Extension-dependent Non-Hybridization) method (WO 2014 / 104818), PCE-IH (PTO Cleavage and Extension-dependent Immobilized Oligonucleotide Hybridization) method (WO 2015 / 008985), and the above patent documents are incorporated herein by reference.

[0110] Additionally, for real-time target detection, a method can be used to detect one or more target nucleic acid sequences using a single type of label by detecting signals at different temperatures. This is disclosed in WO 2015 / 147412, WO 2016 / 093619, and WO 2016 / 093620, all of which are incorporated herein by reference.

[0111] According to one embodiment of the present invention, the amplification reaction may additionally include (i) a probe for detecting a nucleic acid of the target microorganism; and (ii) a probe for detecting a nucleic acid of the Corynebacterium spp. or Cutibacterium spp.

[0112] According to one embodiment of the present invention, the amplification reaction may include 1 to 30, specifically 1 to 25, 1 to 20, 1 to 15, 1 to 10 or 1 to 5 target microorganism nucleic acid detection probes, and may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30 target microorganism nucleic acid detection probes, but is not limited thereto.

[0113] According to one embodiment of the present invention, the primer pair or probe of the nucleic acid of Corynebacterium spp. and Cutibacterium spp. comprises a nucleotide sequence that specifically hybridizes to a nucleotide sequence encoding 16s rRNA or gyrA (DNA gyrase subunit A), or a complementary sequence thereof.

[0114] According to a more specific embodiment of the present invention, the primer pair or probe of the nucleic acid of the Corynebacterium spp. comprises a nucleotide sequence that specifically hybridizes to a nucleotide sequence encoding 16s rRNA, or a complementary sequence thereof, or the primer pair or probe of the nucleic acid of the Cutibacterium spp. comprises a nucleotide sequence that specifically hybridizes to a nucleotide sequence encoding gyrA (DNA gyrase subunit A), or a complementary sequence thereof.

[0115] In a specific embodiment of the present invention, the primer pair or probe of the nucleic acid of the Corynebacterium spp. comprises a nucleotide sequence that specifically hybridizes to the first sequence in the sequence listing, or a complementary sequence thereof, or the primer pair or probe of the nucleic acid of the Cutibacterium spp. comprises a nucleotide sequence that specifically hybridizes to the sixth sequence in the sequence listing, or a complementary sequence thereof.

[0116] The term "specifically hybridizes" as used herein means that two or more molecules interact with each other through covalent or non-covalent bonds, for example, the binding of a single-stranded nucleotide molecule having a single-stranded target sequence and a nucleotide sequence complementary thereto.

[0117]

[0118] Step (d): Determine the validity of the amplification reaction of the target microorganism nucleic acid and determine whether the target microorganism exists (140).

[0119] Finally, the method of the present invention comprises the steps of (d) determining the validity of the amplification reaction of the target microorganism nucleic acid from the result of the amplification reaction of the internal control nucleic acid; and (i) determining whether the nucleic acid of the target microorganism is present in the sample based on the determined validity and (ii) the result of the amplification reaction of the target microorganism nucleic acid.

[0120] According to the present invention, the validity of the amplification reaction result of the target microbial nucleic acid can be determined based on the amplification reaction result of the internal control group among the results of the amplification reaction.

[0121] Typically, the nucleic acid of Corynebacterium spp. or Cutibacterium spp. as an internal control according to the present invention should always be amplified and detected by an amplification reaction, regardless of the presence or absence of the nucleic acid of the target microorganism.

[0122] According to one embodiment of the present invention, when a nucleic acid of Corynebacterium spp. or Cutibacterium spp. is detected as an internal control, it can be determined that the sample collection, nucleic acid extraction, and / or nucleic acid amplification reaction were successfully performed, and the result of the amplification reaction of the target microorganism nucleic acid can be determined to be valid.

[0123] According to one embodiment of the present invention, if the nucleic acid of Corynebacterium spp. or Cutibacterium spp. is not detected as an internal control, it can be determined that a problem occurred in the process of sample collection, nucleic acid extraction, and / or nucleic acid amplification, and the result of the amplification reaction of the target microorganism nucleic acid can be determined to be an invalid result, i.e., invalid.

[0124] In this specification, the term "invalid result" refers to a result in which the detection result cannot be interpreted and is therefore treated as invalid, and the subject of a sample determined to have an invalid result can re-conduct the test for detection from the sample collection stage.

[0125] According to one embodiment of the present invention, if the endogenous internal control nucleic acid is not detected in the amplification reaction result, the result of the amplification reaction of the target microbial nucleic acid can be determined to be invalid.

[0126] According to one embodiment of the present invention, when the endogenous internal control nucleic acid is detected in the amplification reaction result, detection of the target microorganism nucleic acid means the presence (positive) of the target microorganism nucleic acid, and non-detection of the target microorganism nucleic acid in the amplification reaction result means the absence (negative) of the target microorganism nucleic acid.

[0127] According to one embodiment of the present invention, if the endogenous internal control nucleic acid is not detected in the amplification reaction result, the result in which the target microbial nucleic acid is not detected can be judged to be invalid.

[0128] According to one embodiment of the present invention, if the endogenous internal control nucleic acid is not detected in the amplification reaction result, the result in which the target microbial nucleic acid is detected can be judged to be invalid.

[0129] In general, nucleic acid amplification efficiency varies depending on the target nucleic acid, and amplification efficiency also varies within the same tube depending on various factors such as the initial amount of nucleic acid in the sample and the size of the amplified product.

[0130] When the nucleic acid of the target microorganism is present in a relatively large amount compared to the nucleic acid of the internal control, during the amplification process, essential reagents (e.g., polymerase, dNTP, etc.) are consumed while amplifying the nucleic acid of the excess target microorganism, and the amplification efficiency of the nucleic acid of the internal control is relatively reduced, so that the amplification of the nucleic acid of the internal control may not be detected.

[0131] That is, in such cases, even though an endogenous internal control nucleic acid exists in the sample, the nucleic acid of the target microorganism may be amplified, while amplification of the endogenous internal control nucleic acid may not be detected.

[0132] Therefore, according to another embodiment of the present invention, in the amplification reaction result, the endogenous internal control nucleic acid is not detected, and the target microorganism nucleic acid is detected, and it can be determined that the target microorganism nucleic acid exists (is positive).

[0133] According to one embodiment of the present invention, the method of the present invention can be performed by adding an exogenous internal control to a sample collected after sample collection in addition to the internal control described above, and can be performed by including an external positive control and / or an external negative control in the nucleic acid amplification step.

[0134] The above-mentioned external internal control can be used as a control for the nucleic acid extraction process, the above-mentioned external positive control can be used as a control for the nucleic acid amplification process, and the above-mentioned external negative control can be used as a control for sample contamination and non-specific reactions. For example, the external internal control can be added to the sample after sample collection together with the internal control according to the present invention and used. If the external internal control is detected and the internal control according to the present invention is not detected, the sample collection process can be determined to have failed.

[0135] According to another aspect of the present invention, the present invention provides a composition for amplifying a nucleic acid of a target microorganism from a sample collected from the skin using a bacterial nucleic acid selected from the normal skin flora as an endogenous internal control nucleic acid, comprising:

[0136] (i) a pair of primers for amplifying the nucleic acid of the target microorganism; and

[0137] (ii) A pair of primers for amplifying nucleic acids of bacteria selected from the normal skin flora.

[0138] Another aspect of the present invention, "a composition for amplifying a nucleic acid of a target microorganism from a sample collected from the skin using a bacterial nucleic acid selected from the normal skin flora as an endogenous internal control nucleic acid," is manufactured to carry out the above-described aspect, "a method for detecting a nucleic acid of a target microorganism from a sample collected from the skin using a bacterial nucleic acid selected from the normal skin flora as an endogenous internal control nucleic acid," and therefore, the common contents between them are omitted in order to avoid excessive complexity of the present specification.

[0139] According to another aspect of the present invention, the present invention provides a composition for amplifying a nucleic acid of a target microorganism from a sample collected from the skin using a nucleic acid of Corynebacterium spp. or Cutibacterium spp. as an endogenous internal control nucleic acid, comprising:

[0140] (i) a pair of primers for amplifying the nucleic acid of the target microorganism; and

[0141] (ii) A pair of primers for amplifying nucleic acids of Corynebacterium spp. or Cutibacterium spp.

[0142] The "composition for amplifying a nucleic acid of a target microorganism from a sample collected from the skin using a nucleic acid of Corynebacterium spp. or Cutibacterium spp. as an endogenous internal control nucleic acid", which is another embodiment of the present invention, is manufactured to carry out the "method for detecting a nucleic acid of a target microorganism from a sample collected from the skin using a nucleic acid of Corynebacterium spp. or Cutibacterium spp. as an endogenous internal control nucleic acid", and therefore, the common contents between them are omitted in order to avoid excessive complexity of the present specification.

[0143] The composition of the present invention described herein may optionally include reagents necessary for performing a target amplification reaction (e.g., a PCR reaction), such as a buffer, a DNA polymerase cofactor, and deoxyribonucleotide-5-triphosphate. Optionally, the composition of the present invention may also include various polynucleotide molecules, a reverse transcriptase, various buffers and reagents, and an antibody that inhibits DNA polymerase activity. The composition may also include reagents necessary for performing positive and negative control reactions. The optimal amount of reagents to be used in a particular reaction can be readily determined by one of ordinary skill in the art having learned the disclosure herein. Typically, the composition of the present invention is manufactured in a separate package or compartment containing the aforementioned components.

[0144] The features and advantages of the present invention are summarized as follows:

[0145] (a) In a method for detecting a nucleic acid of a target microorganism from a sample collected from skin according to the present invention, a nucleic acid of Corynebacterium spp. or Cutibacterium spp. is used as an endogenous internal control nucleic acid.

[0146] (b) The internal control according to the present invention can be used as an internal control in a sample collection process, an internal control in a nucleic acid extraction process, and an internal control in a nucleic acid amplification process.

[0147] (c) The internal control according to the present invention exists in the sample from the beginning, so there is no hassle of having to add the internal control separately after the sample collection process.

[0148] (d) According to the present invention, by using the nucleic acid of Corynebacterium spp. or Cutibacterium spp. as an internal control, the presence or absence of a target microbial nucleic acid in a sample collected from the skin can be detected with high accuracy by minimizing false negative and false positive judgments.

[0149] Figure 1 is a flowchart of processes for implementing the method of the present invention according to one embodiment of the present invention.

[0150]

[0151] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0152]

[0153] Example

[0154] Example 1: Confirmation of the detection rate of Corynebacterium tuberculostearicum from samples collected from the skin.

[0155] The present inventors confirmed that the nucleic acid of Corynebacterium tuberculostearicum, a bacterium selected from normal skin flora, can be used as an endogenous internal control nucleic acid in a method for detecting target microorganisms from a sample collected from the skin using nucleic acid amplification.

[0156] To this end, the nucleic acid detection rate of Corynebacterium tuberculostearicum was confirmed from samples collected from multiple human skins, and compared with the detection rates of Human beta globin (HBB) and Human Bacteroides spp. (HuBac) genes, which have been used as endogenous internal controls.

[0157] Example 1-1: Preparation of oligonucleotides

[0158] A nucleic acid encoding 16s rRNA was used as the target nucleic acid of Corynebacterium tuberculostearicum (Table 1). The detection of the nucleic acid was performed using TOCE, which can detect multiple targets by utilizing signals generated by dimers formed depending on the presence of the target nucleic acid sequence. TM Technology (WO 2012 / 096523) was used.

[0159] The sequences of the primer pairs, PTO (Probing and Tagging Oligonucleotide) and CTO (Capturing and Templating Oligonucleotide), for detecting the target nucleic acid encoding the 16s rRNA of Corynebacterium tuberculostearicum are shown in Table 2. HBB is a primer pair for detecting HBB (Allplex TM H. pylori & ClariR Assay product) and PTO and CTO designed based on the above primer pair were used. HuBac was used as a primer pair for HuBac detection (Allplex TM Entero-DR Assay product) and PTO and CTO designed based on the above primer pair were used.

[0160] 서열목록설명서열(5’-3’)1Corynebacterium tuberculostearicum의 16s rRNA를 코딩하는 뉴클레오타이드 서열의 일부AGGCGGCGACGGGTAGCCGGCCTGAGAGGGTGGACGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGGGGGATGACGGCCTTCGGGTTGTAAACTCCTTTCGCTAGGGACGAAGCTTTTTGTGACGGTACCTAGATAAGAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTGTCCGGAATTACTGGGCGTAAAGGGCTCGTAGGTGGTTTGTCGCGTCGTCTGTGAAATTCCGGGGCTTAACTCCGGGCGTGCAGGCGATACGGGCATAACTTGAGTACTGTAGGGGTAACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGATGGCGAAGGCAGGTTACTGGGCAGTTACTGACGCTGAGGAGCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGGTGGGCGCTAGGTGTGAGGGTCTTTTCACGACTTTCGTGCCGTAGCTAACGCATTAAGCGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATACACCGGATCGGGCTAGAGATAGTCTTTCCCTTTGTGGCTGGTGTACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCTTATGTT

[0161]

[0162] List of normal skin flora sequences Oligonucleotide sequences (5'-3') Corynebacterium tuberculostearicum 2 Forward primer TACGGGCATAACTTGAGTACIIIIIGGGTAACTG 3 Reverse primer TTCTTCGCGTTGCATCGIIIIIATCCACATG 4 PTOCCATGCGCACTAGAGGGTCTTTTCACGACTTTCGT 5 CTO[BHQ-1]TTTTTTTATTATT[iCy5_T]ATTTTTTGACTCTAGTGCGCATGG[C3 spacer]

[0163]

[0164] Example 1-2: Comparison of detection rates of Corynebacterium tuberculostearicum, HBB, and HuBac

[0165] For target microorganism testing from skin samples, eSwab (Cat. No. 480CE; Copan) was used to collect skin samples from each site (2 axillary samples, 2 groin samples, 3 axillary and groin (mixed) samples). Nucleic acid extraction was performed using the automated extraction equipment Seegene NIMBUS (Cat. No. 65415-03) and the extraction reagent STARMag 96X4 Universal Cartridge kit (Cat No. 744300.4.UC384). 300 μl of sample was used for nucleic acid extraction, and elution was performed in a volume of 100 μl. The obtained nucleic acid extract was used for real-time polymerase chain reaction.

[0166] TaqDNA polymerase with 5' nuclease activity was used for extension of forward and reverse primers, cleavage of PTO, and extension of CTO.

[0167] To compare the detection rates of the target nucleic acid of Corynebacterium tuberculostearicum and the HBB gene and HuBac gene for nucleic acid extracts obtained from the same sample, three tubes containing 5 μl of the same nucleic acid extract were prepared. The first tube (Tube 1) contained 4 pmoles of the forward primer (Sequence 2), 4 pmoles of the reverse primer (Sequence 3), 3 pmoles of the PTO (Sequence 4), and 2 pmoles of the CTO (Sequence 5) for amplifying the target nucleic acid encoding the 16s rRNA of Corynebacterium tuberculostearicum, and the second tube (Tube 2) contained the same amount of the oligonucleotide for detecting Corynebacterium tuberculostearicum and the oligonucleotide for detecting HBB (forward and reverse primers, PTO, CTO). The third tube (tube 3) contained HuBac oligonucleotides (8 pmoles of forward primer, 8 pmoles of reverse primer, 8 pmoles of PTO, and 2 pmoles of CTO). 5 μl of 4X enzyme mixture [finally 3.2 mM dNTPs, 14 mM MgCl2, and 4 U Taq DNA polymerase] and 5 μl of RNase-free water were added to each of the three tubes to prepare a final volume of 20 μl of reaction mixture. Real-time PCR was performed using the reaction mixture prepared above. The tube containing the reaction mixture was placed in a real-time thermocycler (CFX96, Bio-Rad) and denatured at 95°C for 15 min, followed by 45 cycles of 95°C for 10 sec, 60°C for 15 sec, and 72°C for 10 sec. Signal detection was performed at 60°C for each cycle, and the results are summarized in Tables 3 to 7 below.

[0168] As can be seen in Tables 3 to 7 below, eSwab TMThe detection rates of Corynebacterium tuberculostearicum in skin specimens collected by site using the method were 100% for axillary specimens, 100% for groin specimens, and 100% for axillary and groin (mixed) specimens. The detection rates of HBB were 50% for axillary specimens, 100% for groin specimens, and 100% for axillary and groin (mixed) specimens. The detection rates of HuBac were 0% for axillary specimens, 100% for groin specimens, and 100% for axillary and groin (mixed) specimens. In addition, the average Ct value of Corynebacterium tuberculostearicum was 30.01 for axillary specimens, 30.55 for inguinal specimens, and 29.83 for axillary and inguinal (mixed) specimens. HBB had an average Ct value of 41.35 for axillary specimens, 35.62 for inguinal specimens, and 32.23 for axillary and inguinal (mixed) specimens. HuBac had no detection in axillary specimens, an average Ct value of 37.40 for inguinal specimens, and an average Ct value of 35.15 for axillary and inguinal (mixed) specimens, confirming that the average Ct value of Corynebacterium tuberculostearicum was lower than that of HBB and HuBac.

[0169] These results indicate that the nucleic acid of Corynebacterium tuberculostearicum has a superior detection rate at a more stable level (i.e., a lower Ct value than HBB and HuBac) than the genes of HBB and HuBac as an internal control, indicating that the nucleic acid of Corynebacterium tuberculostearicum can be more useful as an endogenous internal control than the HBB gene and HuBac gene, which are commonly used as internal controls.

[0170] Sample Number Ct Value Tube 1 Tube 2 Tube 3 Corynebacterium tuberculostearicum HBB HuBaceSwab_1_Axillary Specimen 32.3341.35N / AeSwab_2_Axillary Specimen 27.87N / AN / A

[0171] Sample Number Ct Value Tube 1 Tube 2 Tube 3 Corynebacterium tuberculostearicum HBB HuBace Swab_1_Inguinal Specimen 31.8938.1136.41 eSwab_2_Inguinal Specimen 29.233.1338.38

[0172] Sample Number Ct Value Tube 1 Tube 2 Tube 3 Corynebacterium tuberculostearicum HBBHuBaceSwab_1_Axillary and Inguinal (Mixed) Specimen 30.3631.4934.92eSwab_2_Axillary and Inguinal (Mixed) Specimen 29.3032.4834.27eSwab_3_Axillary and Inguinal (Mixed) Specimen 29.8332.7236.26

[0173] Sample Number Detection Rate (%) Tube 1 Tube 2 Tube 3 Corynebacterium tuberculostearicum HBBHuBaceSwab_Axillary specimen 2 / 2 (100%) 1 / 2 (50%) 0 / 2 (0%) eSwab_Inguinal specimen 2 / 2 (100%) 2 / 2 (100%) 2 / 2 (100%) eSwab_Axillary and Inguinal (mixed) specimen 3 / 3 (100%) 3 / 3 (100%) 3 / 3 (100%)

[0174] The above detection rate represents the ratio of the number of detected signals to the total number of samples.

[0175] Sample Number Ct Value Tube 1 Tube 2 Tube 3 Corynebacterium tuberculostearicum HBB HuBaceSwab_Axillary specimen Detected Ct range 27.87-32.33 41.35 N / A Mean Ct 30.10 41.35 N / AeSwab_Inguinal specimen Detected Ct range 29.2-31.89 33.13-38.11 36.41-38.38 Mean Ct 30.55 35.62 37.40eSwab_Axillary and Inguinal (mixed) specimen Detected Ct range 29.3-30.36 31.49-32.7 2 34.27-36.26 Mean Ct 29.8 332.23 35.15

[0176]

[0177] Example 2: Confirmation of the detection rate of Cutibacterium acnes from samples collected from the skin.

[0178] The present inventors confirmed that the nucleic acid of Cutibacterium acnes, a bacterium selected from normal skin flora, can be used as an endogenous internal control nucleic acid in a method for detecting target microorganisms from a sample collected from the skin using nucleic acid amplification.

[0179] To this end, the nucleic acid detection rate of Cutibacterium acnes was confirmed from samples collected from multiple human skin samples.

[0180] Example 2-1: Preparation of oligonucleotides

[0181] A nucleic acid encoding DNA gyrase subunit A (gyrA) of Cutibacterium acnes was used as the target nucleic acid (Table 8). The detection of the nucleic acid was performed using TOCE, which can detect multiple targets by utilizing a signal generated by a dimer formed depending on the presence of the target nucleic acid sequence. TM Technology (WO 2012 / 096523) was used.

[0182] The sequences of the primer pairs, PTO (Probing and Tagging Oligonucleotide) and CTO (Capturing and Templating Oligonucleotide), for detecting the target nucleic acid encoding gyrA of Cutibacterium acnes are shown in Table 9.

[0183] Sequence List Description Sequence (5'-3') Part of the nucleotide sequence encoding gyrA of Cutibacterium acnes GTGACGTGCGTGCCGTCATCGACATGGAAGAGGACAAGAAGGGACGCCAGTGCCTGGTCGTCACCGAGTTGCCTTATATGTGCAACCCGGACAACCTCGCCACCAAGATCGCCGACCTGGTGAACTCCGGTCGCATCAACGGTATCGCCGACATCCGTGACGACTCCTCAGCCCGTACTGGTCAGCGTTTAGTCATCGTCCTCAAGCGTGACGCTCAGCCGCGTGTCGTCATGAACAACCTGTACAAGCACACGGCTTTGCA

[0184] List of normal skin flora sequences Oligonucleotide sequences (5'-3') Cutibacterium Acnes 7 Forward primer GTGACGATGCTGCGTGCCIIIIICGACATGGAA 8 Reverse primer TGCAAAGCCGTGTGCIIIIICAGGTTGTTC 9 PTOCCGATGTGTTGGAGCCCGTACTGGTCAGCGTYTA 10 CTO [BHQ-1] ATTTTTTTTTTAT [iQS670_T] TATTATTCAGCTCCAACACATCGG

[0185]

[0186] Example 2-2: Confirmation of the detection rate of Cutibacterium acnes

[0187] For target microorganism testing from skin samples, eSwab (Cat. No. 480CE; Copan) was used to collect skin samples from each site (3 axillary samples, 3 groin samples, 3 axillary and groin (mixed) samples). Nucleic acid extraction was performed using the automated extraction equipment Seegene NIMBUS (Cat. No. 65415-03) and the extraction reagent STARMag 96X4 Universal Cartridge kit (Cat No. 744300.4.UC384). 300 μl of sample was used for nucleic acid extraction, and elution was performed in a volume of 100 μl. The obtained nucleic acid extract was used for real-time polymerase chain reaction.

[0188] TaqDNA polymerase with 5' nuclease activity was used for extension of forward and reverse primers, cleavage of PTO, and extension of CTO.

[0189] Tubes containing 5 μl of target nucleic acid extract of Cutibacterium acnes were prepared for nucleic acid extracts obtained from the same sample. Tube 1 contained 4 pmoles of forward primer (SEQ ID NO: 7), 4 pmoles of reverse primer (SEQ ID NO: 8), 3 pmoles of PTO (SEQ ID NO: 9), and 2 pmoles of CTO (SEQ ID NO: 10) for amplification of the target nucleic acid encoding gyrA of Cutibacterium acnes. 5 μl of 4X enzyme mixture [finally 3.2 mM dNTPs, 14 mM MgCl2, and 4 U Taq DNA polymerase] and 5 μl of RNase-free water were added to the tube to prepare a reaction mixture to a final volume of 20 μl. Real-time PCR was performed using the reaction mixture prepared above. The tube containing the above reaction mixture was placed in a real-time thermocycler (CFX96, Bio-Rad) and denatured at 95°C for 15 minutes, followed by 45 cycles of 95°C for 10 seconds, 60°C for 15 seconds, and 72°C for 10 seconds. Signal detection was performed at 60°C for each cycle, and the results are summarized in Tables 10 to 14 below.

[0190] As can be seen in Tables 10 to 14 below, eSwab TM The detection rates of Cutibacterium acnes in skin samples collected from each site were 100% for axillary samples, 100% for groin samples, and 100% for axillary and groin (mixed) samples. In addition, the average Ct value of Cutibacterium acnes for axillary samples was 35.43, for groin samples was 34.73, and for axillary and groin (mixed) samples was 32.89.

[0191] These results show that the nucleic acid of Cutibacterium acnes can also be used as an endogenous internal control.

[0192] Sample Number Ct Value Tube 1 Cutibacterium acnes Swab_1_Axillary Specimen 35.70 eSwab_2_Axillary Specimen 36.51 eSwab_3_Axillary Specimen 34.09

[0193] Sample Number Ct Value Tube 1 Cutibacterium acnes Swab_1_Inguinal Specimen 36.15 eSwab_2_Inguinal Specimen 34.87 eSwab_3_Inguinal Specimen 33.16

[0194] Sample Number Ct Value Tube 1 Cutibacterium acnes Swab_1_Axillary and Inguinal (Mixed) Specimen 35.18 eSwab_2_Axillary and Inguinal (Mixed) Specimen 30.48 eSwab_3_Axillary and Inguinal (Mixed) Specimen 33

[0195] Sample Number Detection Rate (%) Tube 1 Cutibacterium acnes Swab_Axillary specimen 3 / 3 (100%) eSwab_Inguinal specimen 3 / 3 (100%) eSwab_Axillary and Inguinal (mixed) specimen 3 / 3 (100%)

[0196] The above detection rate represents the ratio of the number of detected signals to the total number of samples.

[0197] Sample Number Ct Value Tube 1 Cutibacterium acnes Swab_Axillary Specimen Detected Ct Range 34.09-36.51 Mean Ct 35.43 eSwab_Inguinal Specimen Detected Ct Range 33.16-36.15 Mean Ct 34.73 eSwab_Axillary and Inguinal (Mixed) Specimen Detected Ct Range 30.48-35.18 Mean Ct 32.89

[0198]

[0199] Example 3: Use of Corynebacterium spp. as an internal control

[0200] The present inventors detected nucleic acids of Corynebacterium spp., bacteria selected from the normal skin flora as an internal control, along with nucleic acid detection of skin microorganisms.

[0201] The present inventors detected Acinetobacter baumannii as a skin microorganism, and used the oligonucleotide included in the Allplex™ MDRO Assay product (Seegene Inc, Korea) as an oligonucleotide for detecting Acinetobacter baumannii nucleic acid, and used the same oligonucleotide as used in Example 1 as an oligonucleotide for detecting internal control nucleic acid.

[0202]

[0203] Example 3-1. Use of Corynebacterium tuberculostearicum as an internal control

[0204] To ensure objectivity and clarity, this experiment used skin samples previously confirmed to be positive for Acinetobacter baumannii using aerobic culture and MALDI-TOF mass spectrometry, which are recognized as standard methods for detecting drug-resistant microorganisms. The samples were cultured for 24 hours at 37°C under aerobic conditions using CHROMagar Acinetobacter medium for the selective isolation and identification of Acinetobacter bacteria.

[0205] Extraction of nucleic acids from the collected skin samples was performed using an automated extraction device, Microlab NIMBUS IVD (Cat. No. 65415-02, Hamilton) and an extraction reagent, STARMag 96X4 Universal Cartridge Kit (Cat. No. 744300.4.UC384, Seegene Inc.). 300 μl of each sample was used for nucleic acid extraction, and elution was performed in a volume of 100 μl. The obtained nucleic acid extracts were used for real-time polymerase chain reaction.

[0206] TaqDNA polymerase with 5' nuclease activity was used for extension of forward and reverse primers, cleavage of PTO, and extension of CTO.

[0207] For each of 5 μl of nucleic acid extracts obtained from samples in which the presence of Acinetobacter baumannii was confirmed and samples in which the presence of Acinetobacter baumannii was not confirmed, (i) 4 pmoles of a forward primer (sequence number 2), 4 pmoles of a reverse primer (sequence number 3), 3 pmoles of PTO (sequence number 4), and 2 pmoles of CTO (sequence number 5) for amplifying a target nucleic acid encoding 16s rRNA of Corynebacterium tuberculostearicum, and (ii) oligonucleotides for detecting Acinetobacter baumannii nucleic acids (forward and reverse primers, PTO, CTO) in the same amount as the oligonucleotides for Corynebacterium tuberculostearicum were added, and 4X enzyme mixture [finally 3.2 mM dNTPs, 14 mM MgCl2, and 4 U Taq DNA polymerase] A reaction mixture was prepared by adding 5 μl of RNase-free water to a final volume of 20 μl. Real-time PCR was performed using the reaction mixture prepared above. The tube containing the reaction mixture was placed in a real-time thermocycler (CFX96, Bio-Rad) and denatured at 95°C for 15 minutes, followed by 45 cycles of 95°C for 10 seconds, 60°C for 15 seconds, and 72°C for 10 seconds. Signal detection was performed at 60°C and 72°C for each cycle, and the results are summarized in Table 15 below:

[0208] Sample Acinetobacter baumanniiCorynebacterium tuberculostearicumAcinetobacter baumannii positive sample20.2331.23Acinetobacter baumannii negative sample--

[0209]

[0210] As shown in Table 15, in the samples in which the presence of Acinetobacter baumannii was confirmed, both the nucleic acid of Acinetobacter baumannii (Ct value 20.23) and the nucleic acid of Corynebacterium tuberculostearicum (Ct value 31.23) were detected. On the other hand, in the samples in which the presence of Acinetobacter baumannii was not confirmed, neither the nucleic acid of Acinetobacter baumannii nor the nucleic acid of Corynebacterium tuberculostearicum was detected. If the internal control is not detected, it means that a problem occurred in the sample collection process, nucleic acid extraction process, or nucleic acid amplification process. When the sample was subjected to aerobic culture, it was confirmed that no bacteria grew on the medium. This means that the sample collection was not performed normally, and since the internal control nucleic acid was not detected, the target nucleic acid detection result can be judged as an invalid result.

[0211] These experimental results demonstrate that Corynebacterium tuberculostearicum nucleic acid can be useful as an endogenous internal control in the detection process of Acinetobacter baumannii.

[0212]

[0213] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

A method for detecting a nucleic acid of a target microorganism from a sample collected from the skin using a nucleic acid of Corynebacterium spp. or Cutibacterium spp. as an endogenous internal control nucleic acid, comprising the following steps: (a) A step of collecting and preparing a sample from the skin; (b) a step of performing an amplification reaction of nucleic acid in the sample using (i) a pair of primers for amplifying nucleic acid of a target microorganism; and (ii) a pair of primers for amplifying nucleic acid of Corynebacterium spp. or Cutibacterium spp., which is an endogenous internal control nucleic acid; (c) a step of detecting the result of the amplification reaction; and (d) determining the validity of the amplification reaction of the target microorganism nucleic acid from the result of the amplification reaction of the internal control nucleic acid; and (i) determining whether the nucleic acid of the target microorganism is present in the sample based on the determined validity and (ii) the result of the amplification reaction of the target microorganism nucleic acid. A method according to claim 1, characterized in that the Corynebacterium spp. and Cutibacterium spp. are normal skin flora. A method according to claim 1, characterized in that the sample of step (a) is collected from moist sites, dry sites or oily sites of the skin. A method according to claim 3, characterized in that the sample of step (a) is collected from a moist area of ​​the skin. A method according to claim 4, characterized in that the moist area of ​​the skin is an armpit, an antecubital fossa, an umbilicus, an groin, a popliteal fossa, or a sole. A method according to claim 1, characterized in that the preparation of the sample in step (a) further comprises a step of extracting nucleic acid from the sample. A method according to claim 6, characterized in that the nucleic acid of Corynebacterium spp. or Cutibacterium spp. is used as an internal control for (i) the step of collecting a sample, (ii) the step of extracting nucleic acid from the sample, and / or (iii) the step of amplifying the extracted nucleic acid. In claim 7, the method further comprises a step of amplifying nucleic acid in the collected sample using a pair of primers for amplifying nucleic acid of the Corynebacterium spp. or Cutibacterium spp., which is an endogenous internal control nucleic acid, thereby determining whether the collection of the sample is valid or invalid. In claim 7, the method further comprises a step of amplifying nucleic acid in the collected sample using a pair of primers for amplifying nucleic acid of the Corynebacterium spp. or Cutibacterium spp., which is an internal control nucleic acid, thereby determining whether the extraction is valid or invalid. A method characterized in that, in the first paragraph, if the internal control nucleic acid is not detected, the result of the amplification reaction of the target microbial nucleic acid is judged to be invalid. A method according to claim 2, characterized in that the normal flora of the skin is the normal flora of human skin. A method according to claim 1, characterized in that the nucleic acid of Corynebacterium spp. and Cutibacterium spp. comprises a nucleotide sequence encoding 16s rRNA or gyrA (DNA gyrase subunit A). A method according to claim 1, wherein the Corynebacterium spp. is Corynebacterium tuberculostearicum, Corynebacterium simulans, Corynebacterium afermentans, Corynebacterium fastidiosum, or Corynebacterium resisten, or the Cutibacterium spp. is Cutibacterium acnes. A method according to claim 1, characterized in that the detection of the amplification reaction result is performed by a post-detection method or a real-time detection method. A method according to claim 1, characterized in that the amplification reaction additionally comprises (i) a probe for detecting nucleic acid of the target microorganism; and (ii) a probe for detecting nucleic acid of the Corynebacterium spp. or Cutibacterium spp. A method according to claim 15, wherein the primer pair or probe of the nucleic acid of Corynebacterium spp. comprises a nucleotide sequence that specifically hybridizes to the first sequence in the sequence listing, or a complementary sequence thereof, or the primer pair or probe of the nucleic acid of Cutibacterium spp. comprises a nucleotide sequence that specifically hybridizes to the sixth sequence in the sequence listing, or a complementary sequence thereof. A method characterized in that in claim 1, the amplification reaction is performed by a PCR method. A method according to claim 1, wherein the target microorganism is a drug-resistant microorganism, a dermatophyte, or methicillin-resistant staphylococcus aureus (MRSA). A composition for amplifying a nucleic acid of a target microorganism from a sample collected from the skin using a nucleic acid of Corynebacterium spp. or Cutibacterium spp. as an endogenous internal control nucleic acid, comprising: (i) a pair of primers for amplifying the nucleic acid of the target microorganism; and (ii) A pair of primers for amplifying nucleic acids of Corynebacterium spp. or Cutibacterium spp. A composition according to claim 19, wherein the Corynebacterium spp. and Cutibacterium spp. are normal skin flora. A composition according to claim 19, characterized in that the nucleic acid of Corynebacterium spp. or Cutibacterium spp. is used as an internal control for (i) the step of collecting a sample, (ii) the step of extracting nucleic acid from the sample, and / or (iii) the step of amplifying the extracted nucleic acid. A composition according to claim 20, wherein the normal skin flora is the normal skin flora of human skin. A method according to claim 19, wherein the nucleic acid of Corynebacterium spp. and Cutibacterium spp. comprises a nucleotide sequence encoding 16s rRNA or gyrA (DNA gyrase subunit A). A method according to claim 19, wherein the Corynebacterium spp. is Corynebacterium tuberculostearicum, Corynebacterium simulans, Corynebacterium afermentans, Corynebacterium fastidiosum, or Corynebacterium resisten, or the Cutibacterium spp. is Cutibacterium acnes. A composition according to claim 19, characterized in that the composition further comprises (i) a probe for detecting a nucleic acid of the target microorganism and (ii) a probe for detecting a nucleic acid of the Corynebacterium spp. or Cutibacterium spp. A composition according to claim 25, wherein the primer pair or probe of the nucleic acid of Corynebacterium spp. comprises a nucleotide sequence that specifically hybridizes to the first sequence in the sequence listing, or a complementary sequence thereof, or the primer pair or probe of the nucleic acid of Cutibacterium spp. comprises a nucleotide sequence that specifically hybridizes to the sixth sequence in the sequence listing, or a complementary sequence thereof. A method according to claim 19, characterized in that the target microorganism is a drug-resistant microorganism, a dermatophyte, or methicillin-resistant staphylococcus aureus (MRSA).

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